Specific, metabolism-driven transporters in excretory epithelia and barrier tissues play a important role in determining xenobiotic uptake, distribution and excretion. Along with xenobiotic metabolizing enzymes, these transporters are our first line of defense against chemical toxins, but they do not distinguish between toxic chemicals and therapeutic drugs. Recent focus has been on xenobiotic transport at the blood-brain and blood-cerebrospinal fluid (CSF) barriers, where we are identifying and characterizing the transporters present and beginning to explore mechanisms that regulate expression and function. The primary structure responsible for the blood-brain barrier is the non-fenestrated brain capillary endothelium. Although originally thought to present a passive, anatomical barrier to xenobiotics, it is now clear that multispecific xenobiotic transporters are a critical feature of the barrier. We use isolated brain capillaries from rat, pig and mouse along with confocal microscopy to study the transporters involved and their regulation. Using this system, we have defined a role for the multidrug resistance associated protein isoform2 in barrier function and demonstrated the involvement of both Mrp2 and p-glycoprotein in the low permeability of HIV-protease inhibitors into the CNS. Moreover, we have recently demonstrated in an animal model that inhibition of p-glycoprotein greatly increases both blood to brain transport of the chemotherapeutic, taxol, and its effectiveness against an intracerebrally implanted human glioblastoma. Nothing is known about regulation of xenobiotic transport at the blood-brain barrier even though our ability to treat disorders of the CNS is greatly impaired by the poor transport from blood to brain of a large number of therapeutic drugs. Current studies are focused on in vitro and in vivo regulation of p-glycoprotein expression by the xenobiotic-activated nuclear receptor, PXR and by rapidly acting hormones, e.g., endothelin. The choroid plexus is responsible for removal of potentially toxic xenobiotics and metabolites from the cerebrospinal fluid (CSF). In this tissue our work has focused primarily on identifying and characterizing transporters that mediate concentrative, Na-dependent uptake of anionic xenobiotics and metabolic wastes from CSF. Using selective inhibitors and an Oat3-null mouse model, we have identified Oat3 as one major contributor to organic anion uptake. Imaging studies with fluorescent organic anions indicate concentrative uptake that cannot be accounted for by the transporters known to be present in the tissue. Finally, we are also using confocal imaging to explore for the first time efflux mechanisms at the blood side of the tissue, where transport appears to be driven by membrane potential.